Universal magnetic co ip kit

Co-IP was performed using Universal Magnetic Co-IP Kit (54002, Active Motif). Briefly, cells were collected using ice-cold PBS/inhibitors buffer. Antibody-cell lysate mixture was prepared by adding 400 μg cell extract and 5 μg TLR4 or p38 antibodies in 500 μl Co-IP/wash buffer in microcentrifuge tubes and was incubated for 4 h at 4°C. Magnetic beads were added to each tube and incubated for 1 h at 4°C with rotating, and washed four times with 500 μl Co-IP/wash buffer. Beads were then resuspended in 2 × reducing loading buffer, heated for 5 min at 95°C, run in a 10% SDS-PAGE gel and subjected to western blot.

Co-immunoprecipitation (co-IP) was carried out on GH3 cells treated with either vehicle (DMSO) or 1 μM BαP for 1 h prior to lysis in whole cell lysis buffer. Co-IP was also carried out on GH3 cells transfected with AHR expression vector or Ahr siRNA and lysates where taken 48 h after transfection. Co-IP was carried out using the Universal Magnetic Co-IP kit (Active Motif, Carlsbad, CA, USA) using the manufacturer’s instructions. 500 µg of whole cell lysate was incubated with 2 µg of Ahr antibody (RPT1, Novus Biologicals, Littleton, CO, USA) for 4 h on ice prior to precipitation with magnetic beads. 20 µg of eluted protein was then analysed by standard Western blot for the presence of the precipitated endogenous Ahr (RPT1 antibody) and the co-immunoprecipitated Rb protein using Rb1 antibody (1F8, Novus Biologicals) for detection. Detection was carried out using anti-mouse secondary antibody (IRDye 800CW, Li-Cor Biotechnology, Lincoln, NE, USA) and read on the Odyssey Imaging System (Li-Cor Biotechnology). β-Actin antibody (AC-15, Novus Biologicals) was used to verify the success and specificity of co-immunoprecipitation. Similar protocol was used to verify the success of transfection with AHR vector and knockdown of endogenous Ahr with siRNA.

Immunoprecipitation was performed using Universal Magnetic Co-IP Kit ((Activemotif, 54002), according to the manufacturer’s instructions. Briefly, Cells were lysed with complete whole-cell lysis buffer, and followed by immunoprecipitation (IP) with anti-TFEB monoclonal antibodies and protein-G Sepharose beads. Cell extracts were then subjected to 10% sodium dodecyl sulfate-polyacrylamide gels and western blot analysis using anti-TFEB or phospho-TFEB (Ser211) or HPA or 14-3-3 monoclonal antibodies, followed by secondary peroxidase-conjugated antibodies.
For immunoblotting, cells were lysed with RIPA lysis buffer (Beyotime, P0013) on ice and centrifuged at 4 °C. Next, a BCA Protein Assay Kit (Beyotime, P0012) was used to determine the protein concentration, according to the manual. Subsequently, western blotting assay was conducted as described previously [34 (link)]. The primary antibodies and secondary antibodies for western blot was listed in Supplementary Table S3.

A Universal Magnetic Co-IP Kit (54002, Active Motif, Carlsbad, CA, USA) was used for Co-IP experiments. Three micrograms of LBP antibody (ab169776, Abcam Ltd., Cambridge, United Kingdom) was added to 500 μg of extracted proteins. The mixture was incubated at 4°C for 60 min with gentle mixing. Then, 25 μL of Magnetic Protein G Beads was added and incubated at 4°C overnight. The mixture was centrifuged at 4000 rpm for 30 seconds at 4°C. The supernatant was discarded, and the Co-IP products were washed four times with PBS. After the final wash, the precipitates were resuspended in 20 μL of sample buffer for western blotting assay. Three micrograms of rabbit IgG (A7016, Beyotime Biotechnology, Nantong, China) and no antibody were used instead of anti-LBP antibody for negative control and blank control, respectively.

Co-immunoprecipitation assays were performed using a Universal Magnetic Co-IP Kit (Active Motif, Carlsbad, CA) according to the manufacturer’s protocol. Nr2e3 antibodies from Santa Cruz Biotech (Cat #: sc-292264 and sc-374513) were used for these co-IP assays. Briefly, MCF-7 or HepG2 cells were lysed in co-immunoprecipitation buffer containing protease inhibitor cocktail, 1 M DTT, 5 M NaCl and Igepal CA 630). The lysates were centrifuged for 10 min at 12, 000 rpm at 4 °C and the resulting supernatant that contains ~2 mg of total protein were incubated either with 3 ug of IgG or of primary antibody at 4 °C overnight in the Co-IP buffer. Then, 20 μL of protein G magnetic beads was added to the mixture and the mixture was then incubated at 4 °C for 3 h. The immunoprecipitated protein complexes were washed thrice with Co-IP buffer. After the supernatant was discarded, the antibody/protein complexes were resuspended in 40 μL of loading buffer and boiled for 5 min. The entire sample was separated with SDS-PAGE and assayed by immunoblotting to detect target protein co-immunoprecipitation.